The immune system is the subject of ever increasing scientific scrutiny. Despite the enormous interest in the immune system and the continuously expanding number of investigators, both academic and industrial, a complete understanding of the system continues to remain elusive. One of the major breakthroughs was identification of the interaction between the T-cell receptor and a major histocompatibility complex antigen. The identification that both class I and class II major histocompatibility complex antigens have a cleft which binds a small peptide provided a significant key to understanding T-cell specificity and T-cell restriction.
Despite an increase understanding of the immune system in advance of immunosuppressive therapy, the major barrier to successful transplant engraftment is the allogenic immune response that results in graft rejection or graft versus host disease. Currently available nonspecific immunosuppressive therapies are accompanied by increased risks of infection and a variety of deleterious side affects, including nephrotoxicity, hypertension, hyperlipidemia and bone disease. Even with the current armament of immunosuppressive agents, acute graft rejection and failure to achieve long-lasting graft survival persists.
Besides transplants, there are a number of other indications where suppression of T-cell proliferation and/or activation would provide a therapeutic methodology for treatment. These include autoimmune disorders, cancer, and the like. Therefore, it would be of substantial value to provide methods for identifying agents that can be used in suppressing the proliferation and/or activation of T-cells.
The present invention expands on the identification that peptides comprising at least eight amino acids from residue 65-79 of the .alpha. chain of and MHC Class II antigen (hereinafter the Class II peptides) can be used to block T-cell proliferation, CTL proliferation, differentiation and lysis. Although the activity of such peptides had been shown, the mechanism of action remained unknown.
The present invention identifies the target of the immunosuppressive Class II peptides. Based on this observation, the present invention provides an assay method and target for identifying and developing agents that can be used to block T-cell proliferation and activation, CTL proliferation, differentiation, activation and/or lysis.
In the examples below, it is shown that a peptide comprising residues 65-79 of an MHC Class II protein (Class II peptides) interacts with members of the Proliferating Cell Nuclear Antigen family of proteins (the PCNA proteins). PCNA proteins had previously been shown to be involved in both DNA replication and nucleotide excision repair (Kuriyan et al. J Mol. Bio 234:915-925 (1993)). PCNA had been described as a structural specific endonuclease with five prime to three prime exonuclease activity that shows homology with putative nucleotide excision repair factors including xeroderma pigmontosum complementation G protein group, S. pombe rad 2 and rad 13, and S. cerevisiae RAD 27/YKL510 and RAD 2 (for example, see Robins, et al. J Biological Chemistry 269:28535-28538 (1994), Waga, et al J. Biological Chemistry 269:10923-10934 (1994) and PCT Application WO96/35715.)
In WO96/35715, it was disclosed that PCNA interacts with Fen.sup.1 and p21. The interaction of PCNA I with FEN.sup.1 and/or p21 had been implicated in being responsible for initiating PCNA activity. The '715 application further showed that a fragment of the Fen.sup.1 protein, containing the sequence motif QGRLDxFF, bound to PCNA somewhere within the center loop of PCNA and blocked PCNA/p21 interaction. (For example also see Waga, et al. Nature 369:574-578 (1994)). The interaction of Fen.sup.1 or p21 with the central loop site on PCNA had been described within the art as being important in PCNA function (for example, see PCT Application WO96/35715).
Quite unexpectedly, the present invention is based on the identification that a Class II peptide, that interacts with and binds to a different portion of PCNA, effects the activity of the immune system, a function previously not ascribed to PCNA. As described below, a peptide fragment corresponding to residue 65 through 79 of a MHC Class II protein was shown to bind to the carboxyl (beta sheet) end of PCNA. This binding mediates the CTL inhibitory activity demonstrated for the MHC Class II peptides (for example see U.S. Ser. No. 08/741,530).